Persistent but benign infection with cytomegalovirus (CMV) is a nearly ubiquitous occurrence in most human populations. However, primary infection during pregnancy can lead to serious CNS disease in the developing fetus or newborn infant and reactivated disseminated infection causes retinitis in AIDS patients and life-threatening pneumonitis, or hepatitis and allograft rejection in immunosuppressed bone marrow and heart, lung, kidney, liver and pancreas transplant patients. CMV is also suspected of contributing to chronic vascular disease and obliterative bronchiolitis in transplant patients and possibly also to restenosis and atherosclerosis. Acute disseminated infection can be reduced but not eliminated by Ganciclovir treatment and resistant strains develop. HCMV is one of the largest and most complex of mammalian viruses and despite much progress in the molecular biology and identi_ing important genes of the virus in recent years, a great deal more basic research is needed to understand its cell biology and pathogenesis. In this project, we have undertaken a detailed analysis of the major immediate-early (MIE) genes of HCMV whose two principal gene products IE1 (IE72, UL123) and IE2(IE86, UL122) function to switch the cell into and out of latency and to prepare the cell for efficient productive lytic cycle infection. The focus of these studies encompasses control ofMIE gene expression via the MIE enhancer domain, the role of IE1 in dispersing PML from its associated intra-nuclear bodies, called PML-oncogenic domains (PODs or ND10) and the pleotrophic roles of IE2 in positive and negative transcriptional control, post-translation regulation of cell cycle events and the switch from cellular to viral DNA replication. Specific projects in the present 5-year proposal include: (I) Analysis of the functional consequences of post-transcriptional interactions of IE2 with four potential cell cycle regulatory proteins p21, JAB1, APC2 and MAD2; (II) continued exploration of the role of PML, DAXX, SUMO and the PODs at very early times in the nucleus, including how IE 1 and IE2 target to the PODS and the role of IE 1 in removing SUMO from PML, in PML and SP100 repression, and in facilitating proper IE2 intranuclear localization; and (III) Evaluation of the viral and cellular protein components, functions and temporal interrelationships between four distinct subnuclear domains containing the IE2 protein, including three that are presumed to be MIE enhancer or Ori-Lyt associated forming "IE transcription sites", pre-replication foci" (pre-RF) and mature viral DNA replication compartments (RC).